1. Field of the Invention
The field of the invention is inducing, detecting and modulating nervous system seizure in animal systems.
2. Background of the Invention
Human seizure disorders are a substantial health problem because of the large number of affected individuals and the variety of different syndromes. For example, an estimated 1% of the U.S. population is affected by over 40 different syndromes that make up the epilepsies (Hauser and Hesdorffer 1990; McNamara 1994; Commission 1989). All individuals are potentially vulnerable to seizures; they can occur in anyone following a sufficiently intense insult to the brain (Noebels 1996). Although seizures can occur in most anyone, individuals vary in what constitutes a seizure-inducing stimulus (Walton 1989; Sackheim et al. 1987). Some individuals have high seizure susceptibility such that they suffer spontaneous seizures while others have low susceptibility such that even head trauma or certain brain tumors would not lead to seizures (Walton 1989). Understanding what causes this variation in seizure susceptibility remains a fundamental problem in the study of human seizure disorders.
Aspects of this disclosure were published by Kuebler and Tanouye in J Neurophysiol. 2000 February;83(2):998-1009. Uchida (Nov 8, 1997, Biochim Biophys Acta, 1349, 13-24) reports the cloning of a mammalian ethanolamine kinase homolog of the eas gene product originally described by Pavlidis et al. (1994, Cell 79, 23-33).
The invention provides methods and composition for inducing, detecting and modulating seizure in animal systems. In a particular embodiment, the invention provides methods for inducing seizure in a fly, comprising the steps of (1) electrically stimulating an unanesthetized fly and detecting resultant seizure induction in the fly (2) electrically stimulating a fly with less than 20V and detecting resultant seizure induction in the fly; (3) electrically stimulating a population of wild-type flies and detecting resultant seizure induction in most of the flies and (4) electrically stimulating a population of flies and quantitatively detecting resultant seizure induction in the flies across genotypes or experience. In particular embodiments, the fly (or flies) is immobilized by mechanics, adhesive or vacuum, stimulated with an electrode tip having a diameter less than 20 um, and/or is a bang-sensitive mutant.
Exemplary subject methods for modulating seizure induction in an animal, comprise the steps of changing the activity of a seizure regulator in an animal system; and confirming a resultant change in seizure inducibility of the system, wherein the regulator is a seizure sensitive mutant suppressor or an enhancer not previously associated with seizure. In particular embodiments, the changing step alters the effective amount of the regulator in the system; the changing step comprises contacting the animal system with an effective amount of an anticonvulsive agent; and the animal system is a fly or a mouse.
A particular application of this method involves detecting agents which modulate seizure induction. In particular embodiments of this application, the changing step is preceded by the steps of (1) forming a mixture comprising a seizure regulator and an agent and detecting either binding of the agent to the novel regulator or a change in the binding of the regulator to a binding target; wherein the confirming step confirms that the agent modulates seizure induction in the system, or (2) forming a mixture comprising a gene encoding the novel regulator and an agent under conditions wherein but for the presence of the agent, the gene provides an unbiased expression and detecting an agent-biased expression of the gene, wherein a difference between the unbiased and agent-biased expression indicates that the agent modulates expression of the gene; wherein the confirming step confirms that the agent modulates seizure induction in the system.
The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation.
In particular embodiments, the invention provides methods for inducing seizure in a fly, comprising the steps of electrically stimulating a fly or population of flies and detecting resultant seizure induction. Any convenient method may be used to electrically stimulate the fly or flies, so long as it provides the requisite seizure induction. Generally, the stimulus is applied on an individual basis, wherein the fly is contacted with an electrode, particularly a relatively small tipped electrode having a tip diameter of less than about 50 um, preferably less than about 20 um, more preferably less than about 10 um, most preferably less than about 5 um, with lower limits bound only by construction and use constraints, generally being from between 0.1 and 1 um, see, e.g. Examples below.
The electrical stimulation required to induce seizure will depend on the fly and the manner in which the stimulation is delivered. With electrically sensitive flies, such as bang-sensitive mutants, seizure may be induced with a voltage of less than about 20V, preferably less than about 10V, more preferably less than about 5 V. With wild-type or suppressed mutants, the voltage requirement can range from 20 to 90V. Generally, at least about 0.1 to 1, and frequently from 1-4 volts are required even for the sensitive flies.
In a particular embodiment, the fly is unanesthetized, meaning that the fly is not subject to anesthesia nor post-anesthesia influences at the time of stimulation, as measured by a deviation from its normal quantitative parameters of seizure induction.
In a particular embodiment, the fly is immobilized to. facilitate direct stimulation. Immobilization may be effected by any convenient means, such as by mechanics, adhesive or vacuum.
Preferred methods provide for quantitative detection of seizure induction, particularly permitting the quantitative measurement of differences in parameters of seizure induction across genotypes and experience. Hence, seizure inducibility in examined animals can be compared to a quantitative scale of values of seizure inducibility, wherein the benchmarks are the quantitatively defined seizure-inducibilities of mutant and wild-type animals.
In particular embodiments, a population of flies is stimulated, generally by serially stimulating individual flies of the population. In one embodiment, the flies are wild-type flies and seizure is detected in a significant portion of the population, generally at least 10%, preferably at least 20%, more preferably at least 50%. In another embodiment, seizure is induced in the flies across genotypes or experience. A wide variety of experiences can be thus evaluated in terms of modulating seizure inducibility, including time, exposure to various stresses such as pharmaceutical agents, etc.
The invention provides a number of methods for modulating seizure induction in an animal. In a particular embodiment, the methods comprise the steps of changing the activity of a novel seizure regulator in an animal system and confirming a resultant change in seizure inducibility of the system. The novel regulator is an enhancer or suppressor of seizure induction, as measured by the assays disclosed herein, not previously associated with seizure. Novel suppressors include regulator is a suppressor gene, or expression product thereof, selected from the group consisting of Sh5, slo, netrin, eag, para, Shrko120, shak-B2, mlenapts and ShKS133. These genes and their products, including transcripts and translates, are well established in a variety of species; preferred homologs are Drosophila, mouse and human.
A wide variety of methods may be used to change the activity of the regulator, depending on the nature of the regulator and the animal. For example, the activity of voltage-gated Na+ channels, such as mlenapts and para, may be changed with an effective amount of a predetermined anticonvulsive agent, e.g. pharmaceutically active agents which specifically interact with such channels, such as phenytoin, carbamazepine and lamotrigine. In other embodiments, the regulator may be directly targeted with antibodies or intrabodies. Alternatively, the activity may be indirectly targeted, such as with competitive inhibitors such as dominant negative mutant forms of the regulator. In yet other embodiments, the expression of the regulator may be changed, for example by regulating the expression of the gene encoding the regulator or introducing vectors which increase (e.g. regulator expression constructs) or decrease (e.g. regulator antisense constructs) regulator expression. Similarly, any seizure inducible animal system may be used: widely used models are available for mouse, rat and fly (Drosophila) systems, see e.g. Seyfried TN, et al. (1999, Adv Neurol;79:279-90) for a review of experimental models of multifactorial epilepsies. the EL mouse and mice susceptible to audiogenic seizures.
A particular application of this method involves screening for and detecting agents which modulate seizure induction, encompassing in vitro, cell-based and animal-based screens. For example, the changing step may be preceded by forming a mixture comprising a novel seizure regulator and an agent, and detecting either binding of the agent to the regulator or a change in the binding of the regulator to a binding target; wherein the confirming step confirms that the agent modulates seizure induction in the subsequently employed animal system.
Alternatively, the changing step may be preceded by forming a mixture comprising a gene encoding the novel regulator and an agent under conditions wherein but for the presence of the agent, the gene provides an unbiased expression, and detecting an agent-biased expression of the gene, wherein a difference between the unbiased and agent-biased expression indicates that the agent modulates expression of the gene; wherein the confirming step confirms that the agent modulates seizure induction in the subsequently employed animal system.